Borrelia burgdorferi, the spirochete responsible for Lyme borreliosis, causes disease in humans when transmitted by the bite of infected Ixodes ticks. Ixodes acquire B. burgdorferi as larvae feed on infected mammals;B. burgdorferi survive in the tick midgut over winter. The larvae molt into nymphs in the spring to emerge as the vector that transmits B. burgdorferi to humans. The long-term goal of this project is to determine the molecular mechanisms that B. burgdorferi employ to survive the low temperatures in the tick during winter. Uncovering these survival strategies fulfills the mission of the agency as the proposed experiments will elucidate a key biological property of this pathogen and provide potential targets to reduce B. burgdorferi viability in tick nymphs, and thus reducing the likelihood of human infection. The hypothesis to be tested is that MalQ, an ?-glucanotransferase, produces cryoprotectant disaccharides that are essential for B. burgdorferi to survive at low temperatures and persist in ticks over winter. This hypothesis will be experimentally addressed in specific Aim 1. The first goal will examine the conditions that induce malQ transcript and protein expression. The hypothesis is that low temperature, and possibly tick-related factors, will induce malQ expression. The second goal will be to determine if B. burgdorferi produces trehalose and if production is dependent on malQ. The third goal will be to determine if B. burgdorferi survival at low temperature is malQ-dependent. The fourth and final goals are to examine if B. burgdorferi survival in Ixodes scapularis at low temperature is malQ-dependent. A genetic screen to identify additional factors involved in survival at low temperatures will be the focus of specific Aim 2. To accomplish this aim, the mariner transposon system adapted for B. burgdorferi will be used to saturate the genome with marked mutations. Low temperature growth assays will identify mutants whose survival has been compromised. Genes that have been disrupted by the transposon will be identified and, thus, implicated in growth at low temperatures and survival in the overwintering tick vector. PUBLIC HEALTH RELEVANCE: The incidence of Lyme borreliosis has steadily increased over the past 15 years with over 27,000 new cases reported in the United States in 2007. B. burgdorferi, the causative agent of Lyme disease, resides in the tick midgut throughout the tick's two year life cycle and is transmitted to humans by tick bite. By studying how B. burgdorferi overwinters in the tick we hope to uncover the molecular mechanisms that allow this bacterium to survive at low temperatures and emerge each summer as a problematic human pathogen.